Cardiovascular Research (2022) 118, 1667–1679 INVITED REVIEW

https://doi.org/10.1093/cvr/cvab214

Hooked on heart regeneration: the zebrafish

guide to recovery
Katherine M. Ross Stewart 1*, Sophie L. Walker 1
, Andrew H. Baker 1
,
Paul R. Riley 2, and Mairi Brittan 1
1
Centre for Cardiovascular Science, University of Edinburgh, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK; and 2Department of
Physiology, Anatomy & Genetics, University of Oxford, Sherrington Building, Sherrington Rd, Oxford OX1 3PT, UK

Received 1 March 2021; editorial decision 23 May 2021; accepted 22 June 2021; online publish-ahead-of-print 23 June 2021

Abstract While humans lack sufficient capacity to undergo cardiac regeneration following injury, zebrafish can fully recover
from a range of cardiac insults. Over the past two decades, our understanding of the complexities of both the inde-
pendent and co-ordinated injury responses by multiple cardiac tissues during zebrafish heart regeneration has in-
creased exponentially. Although cardiomyocyte regeneration forms the cornerstone of the reparative process in
the injured zebrafish heart, recent studies have shown that this is dependent on prior neovascularization and lym-
phangiogenesis, which in turn require epicardial, endocardial, and inflammatory cell signalling within an extracellular
milieu that is optimized for regeneration. Indeed, it is the amalgamation of multiple regenerative systems and gene
regulatory patterns that drives the much-heralded success of the adult zebrafish response to cardiac injury.
Increasing evidence supports the emerging paradigm that developmental transcriptional programmes are
re-activated during adult tissue regeneration, including in the heart, and the zebrafish represents an optimal model
organism to explore this concept. In this review, we summarize recent advances from the zebrafish cardiovascular
research community with novel insight into the mechanisms associated with endogenous cardiovascular repair and
regeneration, which may be of benefit to inform future strategies for patients with cardiovascular disease.
䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏

This paper was handled by consulting editor Jeremy D. Pearson.

* Corresponding author. Tel: þ44 (0)131 242 6689; Fax: þ44 (0)131 242 6779, E-mail: k.ross.stewart@ed.ac.uk
C The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.
V
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse,
distribution, and reproduction in any medium, provided the original work is properly cited.
1668 K.M. Ross Stewart et al.

Graphical Abstract

Zebrafish and human injury response linking-diagrams to represent the effects of tissues on one another. Epicardium: light green, Endocardium: dark green,
Vasculature: blue, Myocardium: red, Lymphatics: purple, Inflammation: yellow, and Extracellular Matrix: orange. Zebrafish follow a highly integrated cardiac
regenerative programme involving all cardiac tissues, illustrated above by the Zebrafish Injury Response linking diagram. This is in direct contrast to humans where
ischaemic injury is predominantly followed by inflammation and extracellular matrix deposition, as illustrated in the Human Injury Response linking diagram,
leading to permanent scarring. It is interesting to consider whether a full understanding of the efficient regenerative programme of the zebrafish could inform
future heart regeneration studies in humans (Krzywinski MI, Schein JE, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA. Circos: An information aes-
thetic for comparative genomics. Genome Res 2009; 31:1639-1645).

....................................................................................................................................................................................................
Keywords Zebrafish • Heart • Regeneration • Model • Myocardial infarction
Hooked on heart regeneration
..
1. Introduction
In humans, myocardial infarction (MI) arising from obstruction of the
coronary circulation engenders massive cardiomyocyte loss, leading to
pathological remodelling and cardiac dysfunction.1,2 Patient care and sur-
vival following acute MI have improved substantially3 but the lack of tar-
geted approaches for effective repair leads to poor long-term
heart function, often resulting in deterioration to heart failure.1,2 This is
exacerbated by the poor regenerative capabilities of adult mammalian
heart, where necrotic and apoptotic tissues are replaced by a permanent
non-contractile fibrotic scar. In 2002, Poss et al.4 first reported the re-
markable regenerative capabilities of the zebrafish heart following ven-
tricular amputation. Since then, additional zebrafish heart injury models,
such as cryoinjury,5–7 genetic ablation,8,9 hypoxia,10 and laser-induced in-
jury11 have been developed and the regenerative responses assessed.
Across all zebrafish cardiac injury models, cell death in the injury area is
followed by a rapid proliferative response in the endocardium,12 epicar-
dium,13 myocardium,14–16 and vasculature,17,18 coupled with a complex
temporally controlled inflammatory response.19 During the first 3 weeks
post-injury, debris is cleared from the injury area and replaced by fibrotic
tissue, akin to the mammalian response.20 This fibrotic tissue is subse-
quently degraded and replaced by electrically coupled vascularized myo-
cardium. Though zebrafish are phylogenetically distant from mammals,
there are distinct similarities in the developmental pathways21,22 and
electrophysiology23 of their cardiovascular systems and the common
evolutionary origin of the heart has been clearly illustrated.24 Zebrafish
are especially well suited to cardiovascular studies (reviewed by Kithcart
and MacRae, 201725) as a result of the high similarity between human
and zebrafish cardiac action potentials. Zebrafish have also been used for
high-throughput drug discovery and compound screening for preclinical
toxicity assessments,26,27 resulting in a number of compounds progress-
ing to clinical trials (reviewed by Cully, 201928). Personalized zebrafish
‘avatars’ are additionally gaining traction. Here, patients’ diseases—
particularly cancer or rare diseases—are modelled in the zebrafish to
provide a platform for developing novel combination treatments, identi-
fying predictive biomarkers, and providing insights into potential treat-
ment resistance mechanisms (reviewed by Costa et al., 202029). While
toxicity screens and disease avatars validate the zebrafish as a relevant
translatable model, there remains a lack of evidence for the ‘bench to
bedside’ pathway for pro-regenerative compounds identified in zebra-
fish, and plenty more work must be done to fully understand how the
zebrafish regenerative programme progresses. With that said, their quick
generation time, large clutch sizes, affordability, ease of genetic manipula-
tion, high degree of genetic conservation with humans, and endogenous
regenerative capabilities are rapidly making zebrafish the model of choice
for many heart regeneration studies.
2. Zebrafish cardiac injury models
2.1 Ventricular resection
In this model, 20–25% of the ventricle is removed surgically with fine
scissors,4,30 causing the heart to bleed profusely. The reduced pressure–
volume of the trabeculated zebrafish heart is such that blood clotting is
sufficient to patch the area of injury. There is then transient collagen
deposition, which is eventually resorbed and replaced by healthy con-
tractile cardiomyofibrils. Full recovery of the ventricle is observed by
30–60 days post-injury.4,30 Since ventricular resection involves the
removal of tissue, there is less requirement for clearance of debris, which
1669
.. may account for the lack of scarring in this injury model (Table 1).
.. Instead, ventricular resection is typically accompanied by an accumula-
..
.. tion of apoptotic cells at the border of the injury zone that does not ex-
.. tend further into the wound site.6
..
..
.. 2.2 Cryoinjury
..
.. Three seminal papers published in 2011 described the development of a
.. cardiac cryoinjury model in the zebrafish.5–7 Cryoinjury has since been
..
.. gaining popularity in adult zebrafish heart studies as it most closely
..
.. mimics human MI (Table 1). The pericardial sac is opened and 20–25% of
.. the ventricle is frozen using a pre-cooled cryoprobe. The rapid tissue
..
.. cooling results in the creation of intracellular ice crystals, which are de-
.. structive to the cells. A uniform central region of coagulation necrosis
..
.. then forms at the probe contact site.31 Towards the peripheral border
..
.. zone, there is a region of apoptotic cell death where the temperature
.. was not sufficiently cold to kill the cells but was cool enough to cause ir-
..
.. reparable damage.31 Cryoinjury results in widespread fibrosis, necrotic
.. cell death,7 and rapid enucleation of cardiomyocytes, whilst leaving the
..
.. sarcomeric structure intact.6 Following cryoinjury, the zebrafish ventricle
.. undergoes cardiac remodelling, taking on a rounded appearance with en-
..
.. largement of the injured ventricle and concomitant wall thickening.5,9
..
.. During the first 3 weeks post-cryoinjury, necrotic tissue cell debris is
.. cleared and replaced by transient fibrosis, which is subsequently
..
.. resorbed and exchanged for functional cardiac tissue.6 Importantly, the
.. pathophysiological consequences of cryoinjury are observed in all car-
..
.. diac cell types, which is in contrast to genetic ablation, discussed below.
..
..
.. 2.3 Genetic ablation
..
.. Cardiomyocytes can be genetically manipulated to express toxins or
.. enzymes that catalyse the production of cytotoxic metabolites. This
..
.. causes cell-type-specific tissue ablation (Table 1). The first genetic abla-
.. tion model in larval zebrafish was designed by Curado et al.,8 in 2007,
..
.. where cardiomyocytes were genetically altered to express nitroreduc-
.. tase (NTR). NTR—through its catalysation of metronidazole (Mtz)—
..
.. forms toxins that result in cell death. Wang et al.9 created a similar injury
..
.. model in the adult zebrafish whereby cardiomyocytes conditionally
.. expressed the recombinase-responsive diphtheria toxin chain A. The de-
..
.. struction of a high percentage of cardiomyocytes activated robust injury
.. responses by surrounding cell types including a full immune response.
..
.. Through the control of cardiomyocyte ablation by the addition and re-
..
.. moval of Mtz8 or tamoxifen9 from the fish water, these systems offer
.. highly reproducible temporal control of the injury as well as the ability to
..
.. induce significant damage non-invasively. Interestingly, the massive loss
.. of cardiomyocytes results in disrupted electric conduction in the ventri-
..
.. cle and induces symptoms of heart failure commonly experienced by
.. humans, including fatigue and impaired swimming speed and endurance.9
..
.. However, in this model only a single-cell type is affected and there is a
..
.. likelihood of a non-specific ‘bystander effect’ on neighbouring cell types.
.. Unlike the other injury models explored in this review, the full cohort of
..
.. destroyed cardiomyocytes in the Wang et al.8,9 adult genetic ablation
.. model is fully regenerated after just 30 days. This suggests that the main-
..
.. tenance of other cell types improves regeneration times by weeks, de-
..
.. spite a greater proportion of the muscle being injured (>60% in genetic
.. ablation compared to 20–25% in ventricular resection or cryoinjury). It
..
.. is interesting, however, that the zebrafish myocardium can be damaged
.. to the extent that the fish enters a state resembling heart failure but can
..
. still be rescued by its intrinsic regenerative capacity.9
1670
..
2.4 Hypoxia/reoxygenation
Of course, the model to most closely resemble human MI would be a lo-
calized induction of hypoxia. Unfortunately, no such model currently
exists in the zebrafish. However, in 2013, Parente et al.10 established a
global hypoxia/reoxygenation model (Table 1). While effective in repro-
ducing hypoxia response mechanisms, this model stimulates hypoxia-
induced injury that is not limited to the heart. There is evidence of in-
flammation and apoptosis in other organs and a global activation of hyp-
oxic response pathways. After induction of this injury model, the
zebrafish displays a transient reduction in cardiac function but the injuries
to the heart are not severe enough to be visible histologically.10 It is in-
teresting to note that a degree of hypoxia is actually required to stimu-
late the regenerative response. Jopling et al.15 showed that exposure to
hyperoxia dramatically impeded zebrafish heart regeneration, while hyp-
oxia improved cardiomyocyte proliferation. Although the global hypoxia
model certainly induces the injury/regeneration response, the quest to
establish a localized hypoxic model in the zebrafish heart continues.
2.5 Explant culture
In 2008, Hecker et al.32 performed a functional and histological evalua-
tion of explanted adult zebrafish hearts. They found that hearts that
were sectioned immediately and 1.5 h after isolation were histologically
indistinguishable,32 thus concluding that the hearts remained viable out-
side the body. Over this time, the hearts additionally maintained sponta-
neous contractile properties. This offered evidence that the isolated
zebrafish heart could provide a powerful, yet simple, tool for assessment
of cardiovascular function. In 2010, this system was adapted to evaluate
the role of Pdgf in epicardial regeneration in the adult zebrafish heart.33
Here, injured and control ventricles were extracted and cultured on a fi-
brin gel. Recombinant Pdgfb and its inhibitors were added directly to the
culture medium to create an ex vivo over-expression and rescue model,
showing a role for Pdgf in mediating the stress response after injury.33
Two independent studies have examined the responses of the explanted
heart to isoproterenol32,34—also known as isoprenaline—which is an
epinephrine analogue commonly used in humans for the treatment of
bradycardia. In both studies the heart rate increased, as expected, in re-
sponse to the drug, highlighting the relevance and extended potential of
an explant system that could aid identification of small molecules to ben-
efit the regenerative response.4 To this end, in 2016, Cao and Poss35
published a protocol to culture live zebrafish hearts—both injured and
healthy—for up to 30 days. Like Kim et al., they used this system to inves-
tigate epicardial regeneration and to identify therapeutic targets for heart
disease. This culture system introduces an array of novel research op-
portunities that were previously inaccessible due to the opacity of the
adult fish. Indeed, while zebrafish larvae are celebrated for their transpar-
ency, in the adult zebrafish repair is primarily assessed using histology,
precluding repeat investigations of the same animal. Tissue explants
could therefore provide a more physiologically relevant alternative to
traditional cell culture experiments (Table 1). Potential drawbacks of ex
vivo assays, however, include a loss of circulating factors and inflamma-
tory responses that will undoubtedly impact the regenerative process.
Further, explanted hearts experience declines in function within 72
h.34,36 This can be somewhat alleviated by the introduction of agitation in
culture,35 but this is not compatible with continuous live imaging techni-
ques. To address this, Yip et al., 2020,36 designed a microfluidics device
that significantly reduced structural and functional declines observed in
other culture methods and permitted continuous live imaging for over 4
days. As we continue to observe improvements in this model, it is
K.M. Ross Stewart et al.
.. important to consider the role it may play in reducing the number of
.. in vivo studies that may cause lasting harm or suffering to the fish.
..
..
.. 2.6 Laser injury
..
.. Matrone et al., 2013,11 developed a laser-induced injury method that is
.. less technically challenging than the other more common and more intri-
..
.. cate resection and cryoinjury models with the added bonus of being
.. completely sterile, highly accurate and requiring a smaller timeframe for
..
.. full functional recovery (Table 1). They showed, both functionally and
.. histologically, that this injury method produces an injury of sufficient se-
..
.. verity to induce a full regenerative response.11 This injury model has
.. since been effectively utilized for in vivo mapping of immune cell recruit-
..
.. ment to the heart after injury via live imaging techniques.37
.. Unfortunately, this model relies on the transparency of the larval zebra-
..
.. fish and no adult adaptations have thus far been reported. It is, however,
.. interesting to consider the possibility of combining the continuously im-
..
.. proving explant culture with this promising injury model to create an ex
.. vivo adult regenerative response assay with simultaneous live-imaging
..
.. opportunities.
..
..
..
.. 3. Cardiomyocyte regeneration in
..
..
.. the zebrafish heart
..
.. One of the cornerstones of heart regeneration is the repopulation of
..
.. functional cardiomyocytes into the injury area. This review explores the
.. mechanisms that drive this in the zebrafish and addresses the intercon-
..
.. nectivity of multiple systems within the heart that are required for
.. successful cardiac regeneration. The genetic signalling programme re-
..
.. sponsible for driving zebrafish heart regeneration at least in part recapit-
.. ulates heart development.14,38 Given conservation of many of these
..
.. mechanisms, this supports comparative studies between species to bet-
.. ter understand potential pathways for organ regeneration in humans.
..
.. Therefore, understanding the origin of the cells that contribute to the
.. regenerating myocardium as well as the transcriptional landscape of
..
.. regenerating zebrafish hearts may aid in identifying areas where mamma-
.. lian injury responses fall short. In 2006, the first study aimed at addressing
..
.. the source of regenerative cardiomyocytes in zebrafish revealed that
.. these cells arise from an undifferentiated progenitor cell population.14
..
.. Later, in 2010, Cre/Lox genetic fate-mapping suggested that pre-existing
.. lineage-committed cardiomyocytes were the predominant contributors
..
.. to myocardial regeneration after ventricular resection.15,39 The
..
.. cardiomyocyte-origin of the regenerated myocardium has since been
.. confirmed in multiple zebrafish injury models.7,11,40 Likewise, neonatal
..
.. mouse cardiac regeneration is achieved through the re-entry of cardio-
.. myocytes into the cell cycle rather than through differentiation of cardiac
..
.. progenitors.41 Studies using 14C isotope dating have revealed that human
.. cardiomyocytes continue to undergo proliferation at a very low rate
..
.. (<1% per year) into adulthood.42,43 Though this cell turnover is not suffi-
.. cient to support regeneration, the potential for amplifying the prolifera-
..
.. tive process in the context of regeneration is intriguing. In the zebrafish,
.. proliferating cardiomyocytes undergo a degree of dedifferentiation that
..
.. includes disassembly of sarcomeric structures, reduced expression of
.. mature sarcomeric proteins and concomitant up-regulation of develop-
..
.. mental myosins44,45 as well as changes to cell adhesion molecules and
.. re-entry into the cell cycle.15,39 In line with reports that border zone
..
.. cardiomyocytes dedifferentiate in response to injury,15,45 Koth et al.,
.. 2020,46 showed an up-regulation of developmental myosin heavy
..
. chain 7 (myh7) in the regenerating heart using single-cell RNA-
Hooked on heart regeneration
..
sequencing (scRNAseq). Similarly, a portion of cardiomyocytes from the ..
subepicardial region reactivate cardiomyocyte developmental marker ..
..
gata4 regulatory sequences. These cells preferentially contribute to the ..
regenerating myocardium, showing reactivation of developmental gene ..
..
programs in mature cardiomyocytes.39 This is further supported by the ..
abrogation of injury-induced cardiomyocyte proliferation in response to ..
..
the expression of a dominant-negative form of developmental gata4.47 ..
For a comprehensive overview of the signalling pathways activated dur- ..
..
ing zebrafish cardiac regeneration, the authors recommend González- ..
Rosa et al., 201748 and Pronobis and Poss, 2020.49 ..
..
An essential factor lending itself to the reaction of zebrafish cardio- ..
myocytes to injury is their sustained responsiveness to mitogenic signals ..
..
into adulthood.16,44 Choi et al.50 performed a chemical screen on zebra- ..
fish larvae to identify small molecules that affected cardiomyocyte prolif-
..
..
eration during cardiac development. They identified compounds acting ..
via hedgehog (hh), insulin-like growth factor (igf) and transforming
..
..
growth factor b (tgfb) signalling pathways that were able to influence the ..
rate of developmental cardiomyocyte proliferation. They then examined
..
..
the effects of those compounds on cardiomyocyte proliferation in adults ..
following ventricular resection and found that they contributed similarly
..
..
to myocardial expansion.50 Further, it was shown that these small mole- ..
..
cules could be pharmacologically manipulated to enhance cardiomyo- ..
cyte proliferation during adult cardiac regeneration.50 This supported ..
..
previous reports that igf signal transduction is required for gata4-
expressing cardiomyocyte proliferation and contribution to regenerating
myocardium.51,52 In addition to igf, signalling by secreted growth factors,
such as fibroblast growth factors,14,16,53 platelet-derived growth fac-
tors,33,52,54 and neuregulins (Nrgs)39,55,56 have also been extensively
shown to stimulate cardiomyocyte proliferation (reviewed by Pronobis
and Poss, 202049). These growth factors function via the Ras/MAPK
pathway, which is tightly regulated by the feedback attenuator Dusp6.
Missinato et al., 2018,54 showed that suppression of dusp6 enhanced car-
diac regeneration following ventricular resection. Inactivation of dusp6
by small molecules or gene inactivation increased cardiomyocyte prolif-
eration as well as neovasculogenesis and reduced fibrosis. As with dusp6,
Koth et al.46 recently recognized runx1 as a potential proliferation damp-
ener. They showed that runx1-/- mutants had higher cardiomyocyte pro-
liferation and increased cardiomyocyte survival in response to injury
than wild-type fish.46 This begs the question as to why an efficiently
regenerating species like the zebrafish would express genes like dusp6
and runx1 that may hamper the regenerative process. The answer
may be linked to the increased proliferation and survival of dusp6/ and
runx1/ cardiomyocytes—phenotypes that are often associated with
cancer. It is well known that humans do not suffer from cancers of the
heart, likely owing to the significantly reduced proliferative capacity of
human cardiomyocytes. Likewise, zebrafish do not develop cardiac can-
cers despite maintaining myocardial proliferative potential and this may
be the result of the expression of proliferative inhibitors, such as dusp6
and runx1. Absence of runx1 results in an up-regulation of the Annexin2a
receptor, a phenotype strongly linked to cancerous proliferation.57 Both
runx1 and dusp6 may therefore function as gatekeepers to maintain con-
trol over the proliferative injury response by mitigating an unrestrained
multiplication of cells. The (in)activation of Notch receptors located on
the endocardium and epicardium following ventricular resection signifi-
cantly impacted cardiomyocyte proliferation. This shows that cardio-
myocytes are highly sensitive to fluctuations in signalling levels, and that
the transcriptomic profile following injury must be tightly controlled.58
Notch has also been shown to mediate the inflammatory response and
the successful regeneration of the endocardium.12 Dynamic induction of
1671
the jak1/stat3 pathway in cardiomyocytes is accompanied by cytokine
production and a prolific immune response,47 once again highlighting the
importance of cross-system communication to drive a full and cohesive
regenerative programme. In addition, spatial RNA analysis (tomo-seq) of
the cryoinjured adult zebrafish heart permitted unbiased genome-wide
mapping of region-specific gene expression, highlighting the reactivation
of developmental pathways post-injury, and a requirement for bone
morphogenetic protein signalling for cardiomyocyte proliferation. A
deeper understanding of the transcriptional regulation of regeneration
will be vital for future regenerative medicine strategies to aid repair with-
out abetting a malignant proliferative response.
Importantly, the transcriptional landscape is not governed by signalling
pathways alone; many of the morphological and transcriptional changes
observed are regulated by epigenetic alterations. Indeed, methylation by
chromatin-remodelling factor Brg1 is a requirement for successful
adult zebrafish heart regeneration.59 Transgenic over-expression of a
dominant-negative form of Brg1 abrogated cardiomyocyte proliferation,
which led to retention of the fibrotic scar. Mechanistically, Brg1 acts via
up-regulation following injury to interact with methyltransferase
Dnmt3a. This leads to down-regulation of cell cycle regulator cdkn1c by
methylation of the cdkn1c promoter.59 Similarly, sarcomere and cyto-
skeletal gene expression in proliferative cardiomyocytes during regener-
ation has been linked to methylation of H3K27.60 Inducing a mutation in
.. histone 3—thereby preventing methylation—results in failed cardiac re-
.. generation in adult zebrafish.60 As with Brg1, H3K27me3-mediated gene
..
.. silencing is essential for adult zebrafish heart regeneration. Thorough ex-
.. ploration into epigenetic chromatin remodelling via DNA methylation
..
.. and histone modifications will augment our understanding of the tran-
.. scriptional changes at play during regeneration.
..
.. Post-transcriptional regulation by way of micro-RNAs (miRNAs)
.. should also not be discounted. Eulalio et al., 2012,61 performed a func-
..
.. tional screen that identified 40 miRNAs that enhanced neonatal mouse
.. and rat cardiomyocyte proliferation, two of which—miR199 and
..
.. miR590—were later shown to improve cardiac regeneration in adult
.. mice. The same group later showed that when miR-199 (which is highly
..
.. conserved in zebrafish62) stimulated cardiac repair when exogenously
.. administered to infarcted pig hearts.63 This indicates that stimulation of
..
.. cardiomyocyte proliferation is attainable in large mammals and that
.. miRNAs could be part of the solution. However, persistent expression
..
.. of miR199 resulted in sudden mortality of most of the treated pigs,63
.. underscoring the importance of gaining a fuller understanding of the car-
..
.. diac regenerative programme in order to deliver a controlled targeted
.. repair strategy. In 2016, Crippa et al.64 performed a comparative gene
..
.. and miRNA profiling of the cardiac transcriptome in mice and zebrafish.
..
.. They identified 45 miRNA-dependent networks that were evolutionarily
.. conserved but differentially regulated in the mammalian and teleost
..
.. models. In particular, they found miR-26a—the most abundant miRNA
.. in the zebrafish heart—to be down-regulated following cardiac insult in
..
.. zebrafish while its levels remained unchanged in the mouse after coro-
.. nary ligation. miR-26a negatively regulates a number of cell cycle activa-
..
.. tors and its inhibition was therefore shown to stimulate cardiomyocyte
.. proliferation.64 Microarray analyses by Yin et al.65 identified a similar pat-
..
.. tern in a number of differentially expressed miRNAs during adult zebra-
.. fish regeneration. One of these—miR-133—targets cell cycle factors
..
.. mps1, cdc37, and pa2g4 as well as cell junction components cx43 and
.. cldn5, giving an indication to its mechanism of action in activating the
..
.. proliferative response when down-regulated. Similarly, after ventricular
.. resection miR-99/100 and let-7a/c are down-regulated, resulting in up-
..
. regulation of predicted targeted genes, which are highly evolutionarily
1672
..
conserved in zebrafish, mice, and humans.66 During development,
miR-99/100 and let-7a/c pathway activity is similar between zebrafish
and mammals, but after MI, expression levels remain constant in mice
but are down-regulated in zebrafish. Interestingly, artificial inhibition of
miR-99/100 and let-7a/c in mice after injury—to match zebrafish expres-
sion patterns—promotes cardiomyocyte dedifferentiation and prolifera-
tion, improving cardiac function and reducing scarring.66 miR-101a
expression was also associated with the zebrafish cardiac injury response
mechanism.67 Within the first 3 days after ventricular resection miR-
101a levels were dramatically reduced, corresponding with the onset of
cardiomyocyte proliferation, but highly up-regulated by 7–14 days, likely
to be associated with removal of fibrosis since prolonged suppression of
miR-101a was accompanied by cardiomyocyte proliferation but a failure
to clear the scar tissue.67 It is interesting that in most of these studies,
changes in miRNA expression triggered by injury in the zebrafish are not
mirrored in mammals, which could point to the contribution of miRNA
to the comparatively high regenerative capacity of the zebrafish heart.
Further, the easy administration of synthetic anti-miRNAs in vivo68 may
suggest that miRNAs could provide a new avenue to explore in the pur-
suit of mammalian cardiac regenerative medicine strategies.
A number of other factors merit consideration in zebrafish cardiac re-
generative ability but are beyond the scope of this review. Regenerative
potential of cardiomyocytes can be influenced by their oxidative status
or by stress44 as well as by genes involved in mitochondrial regulation.69
Environmental and systemic factors, such as overcrowding also impact
regeneration44 as well as hemodynamic shear stress (reviewed by Li
et al., 201956), which affects cardiomyocyte and endothelial cell activity
post-injury. Another interesting contribution to cardiomyocyte prolifer-
ation is that of nerves. Mahmoud et al., 2015,70 showed that the inhibition
of cardiac innervation resulted in impaired cardiomyocyte proliferation
and regeneration. It will be interesting in the coming years to gain a fuller
picture of how these systems contribute to the ultimate goal of full
cardiac regeneration.
4. Vascular regeneration
4.1 Coronary neovascularization
Neovascularization has been shown to be essential for myocardial re-
generation in zebrafish6,17,18,71 and it has been postulated that regenera-
tive neovascularization recapitulates larval coronary vessel development.
This is supported by a 2010 study, where adult zebrafish hearts were
treated with a Pdgfr inhibitor to show that pdgf signalling plays a crucial
role in reactivating developmental angiogenic transcription pathways
during regeneration.33 Interestingly, this pdgf-dependent regenerative re-
sponse appears to be conserved across species as epicardium-derived
Pdgfrb knockout mice display defective coronary artery formation.72
Marı́n-Juez et al., 2016,17 used lineage tracing to reveal that the primary
source of regenerated cardiac vasculature in adult zebrafish is
pre-existing coronary vessels. They found that cryoinjury in adult hearts
expressing the endothelial-specific transgene fli1a: GFP resulted in first
vessel sprouting at 15 h post-injury and that these sprouts formed as
GFPþ extensions of vessels saved from the injury. To investigate previ-
ous reports that epicardial cells were contributing to the new vascula-
ture,14 they then repeated the experiment using fish harbouring the
tcf21: DsRed2 transgene, which labels epicardial and epicardial-derived
cells (EPDC), and found no DsRed2þ cells in the newly regenerated vas-
culature at any timepoint. Building on work by Harrison et al.71 showing
that during development migratory vessels express cxcr4a whose
K.M. Ross Stewart et al.
.. expression is later restricted to arteries, Marı́n-Juez et al.17 observed that
.. revascularization of the damaged area follows a signalling pattern that
..
.. mimics the developmental programme. Zebrafish have two Cxcl12-
.. encoding genes—cxcl12a and cxcl12b—and two genes encoding
..
.. receptors—cxcr4a and cxcr4b.73 The migration of cxcr4b-expressing en-
.. dothelial cells along a Cxcl12 gradient is an established pro-angiogenic
..
.. developmental pathway.73 Harrison et al., 2015,71 showed that cxcr4a
.. mutant zebrafish hearts no longer displayed regenerative potential into
..
.. adulthood in response to ventricular resection. Instead, mutant fish
.. formed scar tissue that was not resolved by 120 days post-amputation.71
..
.. Similarly, in 2019, Marı́n-Juez et al.18 used loss- and gain-of-function
.. experiments to show that superficial revascularization in the cryoinjured
..
.. adult zebrafish heart is regulated by epicardial cxcl12/cxcr4 signalling.
.. They showed that cxcl12b is expressed only in the activated epicardium
..
.. lining the injured area following cryoinjury and that genetic and chemical
.. inhibition of Cxcr4a resulted in blockage of superficial coronary revascu-
..
.. larization.18 The cxcl12/cxcr4 pathway has also been implicated in
.. cardiomyocyte regeneration,74 emphasizing the inter-functionality of re-
..
.. generative signals. As with pdgf, cxc signalling appears to be evolutionarily
.. conserved as mouse Cxcr4 and Cxcl12 knockouts also show develop-
..
.. mental vascular defects.75 It is interesting that the expression of cxcl12b
.. 18
.. by the epicardium appears to be induced by hypoxia via Hif1a. Using
.. hypoxia-related mRNA quantification and a hypoxyprobe, it was shown
..
.. that ventricular epicardial cells are extremely hypoxic at 7 days post-
.. injury and that there were significantly reduced cxcl12b mRNA levels in
..
.. Hif1a deficient hearts compared to wild type controls.18 Similarly, in
.. hif1a-/- ventricles there was a significant reduction in proliferating cardiac
..
.. endothelial cell numbers. In contrast, stabilizing Hif1a resulted in
.. increased endothelial cell proliferation and improved cardiac vessel
..
.. sprouting, indicating that through hypoxia—an environment that is
.. innate to ischaemic injuries—cxcl12b is activated in the epicardium to
..
.. drive vascular regeneration. Indeed, hif1a is not the only hypoxia-related
.. pathway that has been implicated in adult zebrafish cardiovascular regen-
..
.. eration. Earlier this year, it was described how nitric oxide—the reduced
.. form of nitrite, which occurs under hypoxic conditions—improved an-
..
.. giogenesis, immune cell recruitment, and cardiomyocyte proliferation
.. following both cryoinjury and ventricular resection.76
..
.. In addition to epicardial cxc-dependent superficial sprouting, Marı́n-
.. Juez et al.17,18,38 also showed that deeper intra-ventricular coronary
..
.. sprouting after cryoinjury was regulated by vegfaa, which is expressed in
.. the activated endocardium. As early as 15 h post-injury, vegfaa expres-
..
.. sion was observed in the endocardium, coinciding with the initiation of
.. revascularization, and this expression persisted for the duration of active
..
.. cardiac endothelial cell proliferation following injury.18 Knockdown of
..
.. vegfaa dramatically impaired intra-ventricular sprouting, while flt1/
.. fish, which exhibit excessive vegfaa signalling, displayed a greater number
..
.. and length of intra-coronary vessels.18
..
..
.. 4.2 Lymphatic regeneration
.. Regeneration of the lymphatic vasculature is another important
..
.. feature of zebrafish heart regeneration, which also tends to follow
.. development-associated patterns.77–80 Failure of the lymphatic system
..
.. to regenerate results in significant scarring and immune cell retention in
.. the zebrafish heart after cryoinjury,71,77 highlighting how the cardiac lym-
..
.. phatic system influences myocardial regenerative capabilities by facilitat-
.. ing clearance of scar tissue and immune cell debris. In 2019, Harrison
..
.. et al.77 performed intramyocardial injection of microspheres and quan-
.. tum dots to visualize lymphatic vessel development; they found that car-
..
. diac lymphatic vessels developed using coronary arteries as a scaffold.
Hooked on heart regeneration
..
This process was mimicked in a regeneration setting77 and vegfc/vegfr371
and cxc/cxcr71,77 mutant fish, which are unable to form cardiac vascula-
ture, also failed to develop functional lymphatic systems. Vieira et al.,
2018,81 were the first to show that resolution of inflammation following
MI in mice is stimulated by the cardiac lymphatic system via Vegfc. This
led to follow-up studies in the zebrafish that have confirmed that in a re-
generative setting cardiac lymphangiogenesis is stimulated by Vegfc to
improve debris clearance from the wound site and when Vegfc ligands
were inactivated in the lymphatic vasculature prior to cryoinjury, there
was an overwhelming increase in mpxþ neutrophils left in the wound site
as late as 2 weeks post-injury.77 Interestingly, lymphatic vessels efficiently
sprout into the injury area following cryoinjury in adult zebrafish hearts
but less so in response to ventricular resection.77 This is possibly due to
the reduced inflammatory response and lack of scarring that tends to ac-
company ventricular resection, negating the necessity for lymphatic re-
generation. Given the requirement for established vasculature before
lymphangiogenesis can begin,77 there is a resulting lag in the timing of
lymphatic regeneration compared to the coronaries (Figure 1).
5. Epicardium and endocardium
The epicardium and the endocardium are two of the first structures to
undergo regeneration in response to injury in zebrafish (Figure 1), pre-
ceding the responses of the myocardium and the coronary vascula-
ture.12,13,46,48,82 They form active scaffolds to provide mechanical and
paracrine support to guide regeneration of the other cardiac tissues.13,18
Immediately after injury, both the epi- and endocardia are activated in an
organ-wide response.
5.1 Epicardium
Given the strong signalling roles fulfilled by the epicardium, it is no
surprise that epicardial ablation results in impeded cardiomyocyte prolif-
eration and delayed cardiac repair.83 The epicardium undergoes
morphological changes in immediate response to injury,13 which are
accompanied by an induction of developmental signalling markers that
are not expressed in uninjured adult zebrafish.6,7,14,50 This includes reti-
noic acid synthesizing enzyme, raldh2, and developmental transcription
factors tbx18 and wt1.6,7,14 The activation of these markers is initially
observed organ-wide, but by 3 days post-injury, expression becomes re-
stricted to the injury area.7,13 There is also an epicardial paracrine release
of developmental signals18,55,74,84 to contribute to cardiomyocyte and
vascular regeneration and up-regulation of known stimulators of cardio-
myocyte proliferation.47
Epithelial to mesenchymal transition (EMT), wherein the epicardium
forms EPDCs, is another essential component of zebrafish heart regen-
eration. The epicardial cells lose their adhesions and accumulate in the
wound where they undergo EMT to create a thickened ‘epicardial cap’
over the injury site.6,33,85,86 EMT markers, snail and twist, are present in
cryoinjured but not sham-operated hearts,33 indicating that EMT is ex-
clusive to an injury setting in the adult heart. Following EMT, EPDCs mi-
grate into the wound area and differentiate into perivascular cells and
myofibroblasts.15,38 Kikuchi et al.87 showed through Cre/Lox genetic
fate-mapping that after injury, EPDCs also contribute to the smooth
muscle of the bulbus arteriosus. In addition to providing a source of para-
crine signalling and acting as a cell reserve, the epicardium is a mediator
for inflammation14,83,86–88 and strongly expresses extra cellular matrix
proteins, such as fibronectin, periostin, collagen I, and collagen XII
1673
.. to modulate the extracellular environment to be optimized for
.. regeneration.13,55
..
..
.. 5.2 Endocardium
..
.. Less is known about the role of the endocardium, though it is clear that
.. it contributes to the structure of the ECM in the regenerating
..
.. heart.12,82,89 Shortly after induction of epicardial regeneration, the endo-
.. cardium begins to proliferate rapidly to form a regenerating sheet on the
..
.. inside of the wound.12 The endocardial cells also begin their injury re-
.. sponse by undergoing major morphological changes. The cells become
..
.. rounded and partially detach from the surrounding myocardium whilst
.. extending actin-rich filopodia-like protrusions to acquire a motile pheno-
..
.. type at 24 h after injury.12,38 These cells are then reported to spread
.. across the injury area and reorganize into a cohesive sheet to form the
..
.. new regenerated endocardium after 9 days with an endocardial prolifer-
..
.. ative peak occurring at 3 days post-injury, just prior to the 7 days
.. post-injury proliferative peak for cardiomyocytes12 (Figure 1). Like the
..
.. epicardium, the endocardium plays an important role in controlling the
.. extracellular matrix (ECM). scRNAseq data from Koth et al.46 in 2020 in-
..
.. dicated that much of the collagen deposition in the wound was found to
.. be adjacent to myh11-expressing endocardial cells and thrombocytes.
..
.. This observation is consistent with previous reports that the endocar-
.. dium proximal to the wound edge up-regulates collagens to contribute
..
.. to the deposition of scar tissue.12,82 This suggests that while endocardial
.. cells retain their original cellular identity during regeneration, they may
..
.. function analogously to myofibroblasts in the way that they contribute
.. to scar tissue deposition. The dual identity of these collagen-expressing
..
.. endocardial cells may suggest that the injury-induced fibrosis observed in
..
.. the zebrafish is driven by a more transient and less differentiated cell
.. population than myofibroblasts.46 It has been postulated that because of
..
.. this, the collagen deposits are less stable and easier to degrade, which
.. contributes to the transient nature of the zebrafish scar tissue.46 This,
..
.. however, is largely speculative and requires further investigation.
..
..
..
.. 6. Inflammatory response
..
..
.. The importance of temporally controlled tissue inflammation and im-
.. mune cell recruitment as an immediate response to injury is well docu-
..
.. mented.19,90–92 It is known that a dampened immune response after
.. cryoinjury strongly impairs cardiomyocyte mitotic activity91,93 and dimin-
..
.. ishes angiogenesis.90,93 Neutrophils and pro-inflammatory cytokines are
.. observed in the wound area as early as 3 h post-injury.51 However, while
..
.. neutrophils play a crucial role in the tissue repair process as first
.. responders, they also generate reactive oxygen species and secrete
..
.. proteases before undergoing apoptosis.94 For this reason, spatial con-
.. finement and timely clearance are critical to prevent further tissue dam-
..
.. age. Circulating macrophages infiltrate as the second wave responders in
..
.. the immune response, peaking at 7 days after injury.93 Canonically, these
.. cells aid in optimizing the extracellular niche by releasing pro-
..
.. inflammatory cytokines, mediating ECM turnover, activating cardiac
.. fibroblasts, and clearing necrotic debris via release of proteolytic
..
.. enzymes and phagocytosis. They are also responsible for the clearance
.. of neutrophils from the injury location.93 Bevan et al., 2019,19 showed
..
.. that tnfaþ/þ macrophage clearance of neutrophil debris also aided the
.. deposition of the collagenous tissue during the early phases after cryoin-
..
.. jury and that tnfa-/- macrophages facilitate scar removal later in the in-
.. flammatory process. Linking with the role of the efficiently regenerating
..
. lymphatic vasculature, elimination of immune cell debris is a key step in
1674 K.M. Ross Stewart et al.

Figure 1 Overview of the cardiac regenerative timeline of the adult zebrafish heart. Top panel shows graphical representation of regenerating structures
in the injury area. The second panel shows relative waves of regeneration to compare activation timings across structures. The bottom panel shows timing
of these events in hours post-injury and days post-injury, citing key references, and highlighting interaction of systems at each time point.

the regenerative process to maintain an extracellular environment that is

.. highlighted in a number of macrophage ablation studies. Early macro-
..
conducive to healing77,81,93 and indeed, lymphatic regeneration is at its .. phage ablation by clodronate liposomes caused reduced collagen depo-
..
highest just after the macrophage peak around 7 days after injury (Figure .. sition at the injury site, while late macrophage ablation resulted in failure
1). A key study by Sim~ oes et al.,92 revealed that macrophages directly .. of scar resolution.19,91,93 There are a number of molecular mechanisms
..
contribute collagen to the ECM following injury—a phenomenon that .. that may mediate this precisely phased inflammatory response
they found to be conserved in both zebrafish and mice. The importance .. including matrix metalloproteases (MMPs),52,95 cxcl8 and ccl2 signalling95
..
of the macrophage response in regulating the ECM has also been . and Toll-like receptor signalling.93 Given that well-timed inflammation
Hooked on heart regeneration 1675

Table 1 Table to show advantages and disadvantages of zebrafish cardiac injury models.

Injury Schematic Advantages Disadvantages References

..............................................................................................................................................................................................................................
Ventricular resection Fast recovery No lymphatic regeneration Poss, Wilson & Keating,
Common in literature Less similar to human MI 20024; Raya et al., 200330
Injury to all cell types Technically challenging
Variable injury size
Open chest model

Cryoinjury Common in literature Long recovery Chablais et al., 20115;

Most similar to human MI Technically challenging González-Rosa et al.,
Injury to all cell types Open chest model 20116; Schnabel et al.,
20117

Genetic ablation Cell-specific study Limited to single-cell type Curado et al., 20078; Wang
Non-invasive Less similar to human MI et al., 20119
Fast recovery
Technically simple

Hypoxia/reoxygenation Reperfusion injury No localized version currently Parente et al., 201310

Non-invasive available
Similar to human MI
Technically simple

Explant culture Fewer ethical concerns Loss of circulating factors Hecker et al., 200832;
No requirement for home of- Declines in heart function Pieperhoff et al., 201434;
fice approval Injury response from Cao and Poss., 201635; Yip
Live imaging extraction et al., 202036

Laser injury Non-invasive Limited to larval stages Matrone et al., 201311; Kaveh
Consistent injury Requirement for specialized et al., 202037
Fewer ethical concerns equipment
No requirement for home of-
fice approval
Live imaging
High N-numbers

aids rather than inhibits regeneration,7,9 remarkably little is currently

.. myocardium. In humans, this is a permanent fibrotic response that results
..
known about the regulation of inflammatory signals in the injured zebra- .. in adverse functional consequences, which can progress to heart fail-
..
fish heart. .. ure.96 Persistence of a fibrotic scar is therefore often cited as the key dif-
... ference between the non-regenerative human response and the efficient
.. recovery of the zebrafish heart. Immediately following cardiac injury in
..
7. ECM remodelling .. the zebrafish, a fibrin clot forms over the injury site, which is rapidly fol-
.. lowed by the accumulation of myofibroblasts. These myofibroblasts are
..
The ECM is a network of proteins responsible for the structural integrity .. responsible for modulating the ECM by expressing proteins, such as col-
of the myocardial tissue that allows for electrical transmission between .. lagens, fibronectin, vimentin, and tenascins and by controlling the electri-
..
the cardiomyocytes. After injury, fibrosis develops at sites of cardiomyo- .. cal coupling and contractility of adjacent cardiomyocytes.5,55,97,98
cyte necrosis in order to preserve the structural integrity of the
.. Importantly, deposition of ECM components also function to alter the
1676
..
tension of the tissue, which can induce signalling via mechanosensitive ..
pathways.56,97 Wang et al., 2013,55 applied a proteomics approach to ..
..
show that fibronectin—a major ECM protein—is deposited by the epi- ..
cardium after cardiac injury in the adult zebrafish. They showed that two ..
..
fibronectin paralogues are expressed in epicardial cells following an in- ..
jury event, while the IntegrinB3 receptor is expressed in surrounding ..
..
cardiomyocytes. Through the integrin receptors, extracellular signals ..
from the fibronectin are transduced, though they do not appear to make ..
..
a direct contribution to cardiomyocyte proliferation during the repair ..
process. Instead, it appears that fibronectin is required for cardiomyo- ..
..
cyte mobilization and integration into the injury zone.55 Similarly, integ- ..
rins on the basal surface on endothelial cells connect them to ECM ..
..
components and act as mechanosensors to influence the development ..
of new blood vessels.56 The observation that macrophages,92 epicar-
..
..
dial,55 and endocardial cells46 also contribute collagen to this tissue chal- ..
lenges the dogma that myofibroblasts are the sole source of scar tissue
..
..
post-cardiac injury and emphasizes the requirement for multi-tissue ..
interactions during regeneration. Due to the persistence of scarring in
..
..
adult mammals, the fibrotic response is often considered to be inhibitory ..
to regeneration, but this has unequivocally been shown not to be the
..
..
case.6,40,82,99 In fact, it is probable that fibrosis has been evolutionarily se- ..
..
lected. Indeed, limiting the fibrotic response during the early phases after ..
injury results in failed regeneration.40,82 ..
..
Molecularly, the expression of key factors to control the intricate ..
scarring process remains largely unexplored. Chablais et al.40 showed ..
..
that part of the balance between scar resolution and persistence is con-
trolled by smad3-dependent tgfb/activin signalling. The expression of type
I receptor alk5b (tgfr1b) is seen in both the cells of the fibrotic scar and
the neighbouring cardiomyocytes. Ligands for these receptors are locally
expressed during cryoinjury thereby activating the tgfb signalling pathway
in both the injury zone and the cardiomyocytes surrounding the trauma.
Blocking this pathway through chemical receptor inhibition resulted in
failure of the heart to regenerate at all. Using this reversible inhibitory ap-
proach, the researchers were able to identify three critical stages of
zebrafish heart regeneration, which include (i) the deposition of a tran-
sient collagen-rich scar, (ii) Tenascin C-associated tissue remodelling at
the infarct border, and (iii) cardiomyocyte proliferation.40 It is interesting
to note that of the three required processes for successful regeneration,
two of them pertain to ECM structural remodelling.
In addition to smad/tgf signalling, it is also clear that many of the com-
ponents of the fibrotic ECM are broken down by MMPs. The action of
MMPs in the injury area helps to control not only ECM turnover, but
also contributes to inflammatory signalling, which in itself modulates the
extracellular environment by clearing necrotic tissue.100 Collagenolytic
activity in the regenerating adult zebrafish heart is primarily regulated by
MMP2 and MMP14a,101 whose activity decreases as the myocardium
regenerates. Expression of the genes coding for MMP2, MMP14a, and
MMP14b begin at 7 days post-injury and last until 14 days post-injury,
suggesting that ECM remodelling occurs in the middle stages of zebrafish
heart regeneration. In a comparison between heart and fin regeneration,
tissue remodelling genes made up the bulk of the gene expression cate-
gories that matched in the two zebrafish regeneration models.52
Comprehensive mining of DNA microarrays and Gene Ontology term
enrichment analyses for regenerating zebrafish hearts by Mercer et al.98
revealed that distinct ECM components and ECM-modifying proteases
are among the most significantly enriched genes in response to local in-
jury. In contrast, data analyses for mammalian cardiac injury models indi-
cated that inflammation and metabolic processes are the most
significantly activated gene groups.98 It is worthy of note that the
K.M. Ross Stewart et al.
zebrafish and mammalian responses to injury do not differ significantly in
the early stages, but where mammals present with prolonged inflamma-
tion and retention of scarring, these phases are transient in the zebrafish.
Elimination of profibrotic cells has been postulated to progress via ap-
optosis102 but TUNEL staining of postnbþ fibroblastic cells reveals that
there is remarkably little cell death following injury. Sánchez-Iranzo et al.,
2018,82 showed using lineage tracing and RNA-sequencing that during fi-
brosis regression, the fibroblasts are not eliminated but instead persist in
the wound site but in a deactivated state. While still present, the postnbþ
cells drastically reduce expression of ECM-associated genes although
col7a1l and col8a2 remain up-regulated from 7 to 60 days post-injury
compared to uninjured control hearts, indicating that while the fibro-
blasts are largely deactivated, they do not fully revert to a homeostatic
baseline expression profile. This serves to highlight how sophisticated
the zebrafish post-injury fibrotic response is and though there is resolu-
tion of the scar in the zebrafish, there remains a careful control of the ex-
tracellular environment for months after the fact.
8. Discussion
Human therapeutic cardiac regeneration is a long sought-after goal, and
the heart is an area of rapid expansion in zebrafish disease modelling,
though the phylogenetic distance between zebrafish and mammals
means that important questions remain to be answered about the trans-
latability of these studies. This review has highlighted the myriad benefits
..
.. of zebrafish as a model. The extensive repertoire of cardiac injuries avail-
.. able for study in the zebrafish invites elegant solutions to a number of dif-
..
.. ficulties posed by other animal models: live imaging capabilities have
.. provided unprecedented levels of in vivo data on the injury response
..
.. mechanisms and cell–cell interactions therein37 and drug screens with
.. N-numbers well into the thousands have enabled low-cost, high-
..
.. throughput drug discovery.27 The Zebrafish Model Organism Database
.. (ZFIN) (https://zfin.org) is available to researchers as a central repository
..
.. for genetic and phenotypic data on zebrafish models of human dis-
..
.. ease.103,104 This encourages open science and high reproducibility.
.. Open-source online databases—like ZFIN—have also aided precision
..
.. genome editing. In this regard, CRISPR/Cas9 has been one of the most
.. important technical advances for zebrafish disease modelling. A major
..
.. caveat that complicates genetic studies in the zebrafish is the duplication
.. of many genes, making the creation of knockdown strains difficult, which
..
.. can confound forward genetic approaches due to potential compensa-
.. tory mechanisms. Advances in RNA and proteomic sequencing at the
..
.. single-cell level will help us to gain a much fuller picture of the transcrip-
.. tional and translational profile of the zebrafish heart at key timepoints af-
..
.. ter injury. This is supported by the constantly expanding database of
.. regulatory miRNAs involved in zebrafish cardiac regeneration, with in-
..
.. sight into the differences in expression levels of conserved targets in
..
.. non-regenerative mammals after injury.64,66 This review has discussed
.. the cohesive inter-system response of the zebrafish heart to injury,
..
.. though there remains plenty to be discovered and the translational bene-
.. fits of these findings remain to be fully proven. A greater degree of col-
..
.. laboration between groups focusing on different cell types will provide a
.. better understanding of inter-tissue interactions during regeneration.
..
.. With the gaining popularity of zebrafish models, researchers await an in-
.. crease in the number of reagents—such as antibodies—suited to zebra-
..
.. fish tissues, the current paucity of which can impede in-depth molecular
.. investigations. Attempts at augmenting human recovery have thus far
..
. tended to rely on cardiomyocyte stimulation alone, but these single-
Hooked on heart regeneration 1677

..
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